Abstract

The number of nodules that form in a legume when interacting with compatible rhizobia is regulated by the plant. We report the identification of a mutant in nodule regulation in Medicago truncatula, like sunn supernodulator (lss), which displays shoot-controlled supernodulation and short roots, similar to sunn mutants. In contrast with the sunn-1 mutant, nodulation in the lss mutant is more extensive and is less sensitive to nitrate and ethylene, resembling the sunn-4 presumed null allele phenotype. Although the lss locus maps to the SUNN region of linkage group 4 and sunn and lss do not complement each other, there is no mutation in the genomic copy of the SUNN gene or in the 15-kb surrounding region in the lss mutant. However, expression of the SUNN gene in the shoots of lss plants is greatly reduced compared with wild-type plants. Analysis of cDNA from plants heterozygous for lss indicates that lss is a cis-acting factor affecting the expression of SUNN, and documented reversion events show it to be unstable, suggesting a possible reversible DNA rearrangement or an epigenetic change in the lss mutant. Assessment of the SUNN promoter revealed low levels of cytosine methylation in the 700-bp region proximal to the predicted transcription start site in both wild-type and lss plants, indicating that promoter hypermethylation is not responsible for the suppression of SUNN expression in lss. Thus, lss represents either a distal novel locus within the mapped region affecting SUNN expression or an uncharacterized epigenetic modification at the SUNN locus.

Symbiosis between two organisms requires cross-species communication, often using molecular signals. For a symbiosis to be evolutionarily successful, there must be a balance of costs and benefits between the two organisms; the tradeoffs made by each to establish the association must be balanced by benefits gained. The interaction of legumes and nitrogen-fixing rhizobia has several such tradeoffs. The plant alters its developmental program to provide a home for the bacteria in specialized structures called nodules. In exchange for this energy-intensive development, the plant receives nitrogen in a biologically usable form from the bacteria. The bacteria also change their developmental program, differentiating into bacteroids, and receive energy and carbon skeletons from the plant in exchange for the nitrogen they provide. The application of molecular genetic techniques to the study of this interaction has yielded many new insights. Because the biological cost to the plant of nodule formation and function is estimated at 12 to 17 g of carbon per gram of nitrogen (Crawford et al., 2000), the host plant tightly regulates the number and positions of the nodules that form. Plants that have lost this regulation through mutation form excessive nodules, resulting in a phenotype termed hypernodulation or supernodulation.

At the molecular level, flavonoids secreted into the soil by the plant induce the expression of rhizobial nod genes, resulting in the synthesis of a modified (substituted) lipochitin oligosaccharide called the Nod factor. A species-specific interaction between the Nod factor and the molecular receptor(s) on the plant root hairs causes a set of physical and genetic changes that allow the bacteria to enter the plant tissue. The root hair grows so as to curl around and entrap the bacteria, forming a structure termed a crozier, or shepherd’s crook. In legumes that maintain persistent nodule meristems, such as alfalfa (Medicago sativa) and pea (Pisum sativum), the plant cells display cytoskeletal rearrangements that accompany cell division and that facilitate the growth of a plant-generated infection thread from the root hair cell to the inner cortical cells. At the same time, genetic reprogramming of the inner cortical cells adjacent to the xylem poles results in the formation of a nodule meristem that will eventually receive the bacteria in intracellular compartments called symbiosomes. Once inside the nodule in symbiosomes, the rhizobia will express the nitrogenase enzyme and fix nitrogen for the plant, while obtaining carbon skeletons across the symbiosome membrane from the plant in return. This symbiosis allows legumes to grow in the absence of available soil nitrogen. The plant regulates this process: in the presence of available soil nitrogen, nodulation is suppressed (for review, see Riely et al., 2006; Stacey et al., 2006; Oldroyd and Downie, 2008).

At present, sunn and skl are the only published supernodulation mutants in M. truncatula. Each is represented by multiple alleles. SUNN encodes a Leu-rich repeat receptor kinase with homology to Arabidopsis (Arabidopsis thaliana) CLAVATA1 (CLV1), although it shows none of the meristem defects of Arabidopsis clv mutants (Schnabel et al., 2005). Orthologs with very similar phenotypes have been isolated from soybean (Glycine max), Lotus japonicus, and pea (Krusell et al., 2002; Nishimura et al., 2002a; Searle et al., 2003). Plants with a mutated SUNN gene have shorter roots and 5- to 10-fold more nodules when compared with wild-type plants (Penmetsa et al., 2003; Schnabel et al., 2005; van Noorden et al., 2006). Several mutations in the SUNN gene have been identified: the mutation in the sunn-1 allele results in an amino acid change in the kinase domain of the protein, sunn-2 is an amino acid change in the Leu-rich repeat region, while the mutation in the sunn-4 allele results in a truncation of the message immediately after the initial signal peptide sequence and is presumed to act as a null mutation with no SUNN protein produced (Schnabel et al., 2005).

The SKL gene encodes a protein with homology to Arabidopsis EIN2 (a component of the ethylene signaling pathway), and the mutants have several ethylene-related phenotypes such as long roots in addition to nodulation defects (Penmetsa et al., 2008): excessive nodule number and loss of spatial control (position relative to xylem) of nodule formation (Penmetsa and Cook, 1997). An ortholog to SKL affecting nodulation in other legumes has yet to be identified, but overexpression in L. japonicus of a dominant negative allele of another Arabidopsis ethylene response gene, ETR1, increases nodule number (Gresshoff et al., 2009).

Supernodulation mutants with no clear ortholog yet in M. truncatula have been identified in other legumes. The astray mutant in L. japonicus results in moderately increased nodulation (2-fold versus the 5- to 10-fold usually associated with hypernodulation). The corresponding gene encodes a basic Leu zipper protein with a ring finger motif similar to Arabidopsis HY5 (Nishimura et al., 2002b). Another L. japonicus supernodulation mutant, klavier, causes pleiotropic effects on flowering time, plant stature, and venation (Oka-Kira et al., 2005). The rdh1 and tml mutants of L. japonicus also display a supernodulation phenotype; grafting experiments have shown that the signal controlling nodule number in these mutants is root derived (Ishikawa et al., 2008; Magori et al., 2009). Mutants described only at the phenotype level include the Phaseolusvulgarisntsn supernodulation mutant (Park and Buttery, 1989) and two pea mutants, sym28, a shoot-controlled nodulator with a fasciation phenotype (Sagan and Duc, 1996), and nod3, a root-controlled supernodulation mutant (Jacobsen and Feenstra, 1984).

Here, we report a novel M. truncatula supernodulation mutant distinct from skl and the Pisum and Lotus mutants described above. The like sunn supernodulator (lss) mutation interacts genetically with sunn, yet it is not a lesion in the SUNN gene proper or in the adjacent regulatory sequences. Nodulation in lss is more extensive than in sunn-1 and is less sensitive to nitrate and ethylene. The expression of the SUNN gene is greatly reduced in lss mutants, and overall, the lss phenotype resembles the phenotype of the sunn-4 presumed null mutation. Results of our analyses are consistent with lss exerting an epigenetic effect on SUNN expression.

RESULTS

The lss Mutation Is Recessive, with Supernodulation and Short-Root Phenotypes Resembling Those of the sunn Mutation

Based on a visual screen for altered nodule number, we identified a spontaneously occurring supernodulation mutant from the Jemalong cultivar (from which the wild-type A17 line was derived), which we designated lss. Following inoculation with rhizobia under nitrogen-limiting conditions, plants carrying the mutation have a 10- to 15-fold increase in nodule number and shorter roots than wild-type plants. When grown in aeroponic conditions side by side with the wild type and sunn-1 and sunn-4 mutants, plants bearing the lss mutation strongly resemble the previously identified sunn-4 mutant (assay done at 10 d post inoculation; Fig. 1; Penmetsa et al., 2003; Schnabel et al., 2005). Both lss and sunn-4 form significantly more nodules than sunn-1.

Nodulation and root phenotypes of the lss mutant. A, Average nodule number of 14-d-old plants grown under aeroponic conditions in the presence or absence of 10 mm nitrate in the form of KNO3 or NH4NO3 (n = 17 to 25 plants per genotype) at 10 d post inoculation (pi). B, Average root length (in mm) of the same plants as in A. Error bars for all graphs indicate se. When compared with the wild type, P < 0.05 by Student’s t test for all comparisons within a treatment in A and the no-nitrate comparison in B.

Consistent with previous work, we observed little to no nodulation on wild-type plants supplied with 10 mm nitrate as either NH4NO3 or KNO3 (Fig. 1A). In contrast, lss mutant plants demonstrate nitrate-tolerant nodulation. When grown aeroponically in the presence of 10 mm ammonium nitrate and compared with sunn-1 and sunn-4 mutant plants, lss mutant plants form more nodules than wild-type or sunn-1 plants and have nodule numbers similar to sunn-4 plants (Fig. 1A).

We measured the root length of nodulated and uninoculated plants. In nitrate-free conditions, nodulated sunn-1, sunn-4, and lss roots grew to only about half the length of wild-type roots (Fig. 1B, black bars). In the presence of 10 mm NH4NO3, all genotypes, including the wild type, displayed the same short-root phenotype when nodulated (Fig. 1B, striped bars). Since supernodulator roots grew the same under both nitrate conditions, we wondered if their short-root phenotype was due to excessive nodule development and could not be further reduced by nitrate. However, when plants were grown under nonnodulating conditions (no rhizobia) and in nonlimiting nitrate, sunn-1 and lss still exhibited the short-root phenotype (Fig. 2; P < 0.001, Student’s t test).

Root length of the lss mutant in the absence of rhizobia. Root length is shown for wild-type, sunn-1, and lss plants grown under aeroponic conditions in the presence of 5 mm NH4NO3 without rhizobia. This concentration of nitrate is not limiting for root growth. Data are from 14-d-old plants (n = 24–25 plants for sunn-1 and A17 and n = 40 plants for lss). Error bars indicate se. Both sunn-1 and lss are significantly different from the wild type (P < 0.001 by Student’s t test).

Grafting experiments revealed that for lss mutants, the genotype of the shoot tissue determines the number of nodules that form (Table I), as was previously shown for the SUNN gene (Penmetsa et al., 2003). Because of the similar nodulation, root length, and shoot-control phenotypes, we named the mutant lss.

Genotypes are listed in columns 1 and 2, and nodulation was measured 11 to 17 d following mending of the graft and inoculation. Results are reported ±sd.

To define the map position of the lss locus, pollen from the wild-type polymorphic mapping ecotype A20 was used in crosses with homozygous lss individuals. All four F1 heterozygotes derived from this cross displayed wild-type nodulation and root length. In a backcross of lss to parental A17, all eight progeny also displayed wild-type nodule number and root length. Both lines of evidence indicate that lss is a recessive allele. F2 progeny of the A20 cross (n = 209) were scored for nodule number 10 d post rhizobial inoculation; 44 plants displayed the supernodulation phenotype, while the remaining 165 individuals showed wild-type nodulation. The phenotypic ratio of 165:44 closely approximates 3:1 (χ2 = 1.73, P < 0.05) and is consistent with the phenotype resulting from a single gene mutation.

lss and sunn-1 Mutants Have Different Responses to Ethylene

Because altered ethylene responses have been documented in previously published M. truncatula supernodulation mutants (Penmetsa and Cook, 1997; Oldroyd et al., 2001; Penmetsa et al., 2008), we tested the response of lss to ethylene. Previously, we had reported that nodulation in both sunn-1 and wild-type A17 was reduced in the presence of 1-aminocyclopropane-1-carboxylic acid (ACC), an ethylene precursor (Penmetsa et al., 2003). We observed here that lss, sunn-4, and wild-type plants appeared less sensitive to ACC inhibition of nodulation than did sunn-1 (Fig. 3A). Our standard growth conditions on plates include 0.1 μm aminoethoxyvinylglycine (AVG) added to the medium to inhibit excess ethylene biosynthesis and buildup on the plate, thus allowing plants to nodulate. Therefore, we tested nodulation on plates with a range of ACC and AVG concentrations that allowed high to low ethylene accumulation. AVG (low ethylene) increased nodulation in all plants but was more effective on wild-type, sunn-4, and lss plants than on sunn-1 plants, again indicating that sunn-1 is more sensitive to ethylene (Fig. 3A). When suppression of nodulation was considered as a percentage of the normal nodulation observed under our standard conditions (0.1 μm AVG), relationships were more complex, but examining a subset of the data from Figure 3A in this manner revealed that the sunn-1 nodulation response is more sensitive to ethylene than that of sunn-4, lss, or the wild type (Fig. 3B). Since this phenotypic distinction could involve differences in either ethylene perception or ethylene generation by the mutants, we measured ethylene production in the mutants versus the wild type after inoculation and compared it with the ethylene-insensitive skl supernodulator, which in our assay overproduced ethylene. Ethylene levels were the same in the wild type, both sunn alleles, and lss, suggesting that any observed difference in ethylene response is related to ethylene perception (Fig. 3C).

Ethylene-related phenotypes of the lss mutant. A, Average nodule number of A17 (wild-type), sunn, and lss plants grown on decreasing concentrations of the ethylene precursor ACC and increasing concentrations of the ethylene biosynthesis inhibitor AVG (n = 5–15 plants per genotype per treatment). Error bars indicate se. B, A subset of the data in A displayed as a percentage of the normal nodule number seen under our standard conditions of 0.1 μm AVG. C, Ethylene content as parts per million per gram fresh weight of inoculated wild-type, sunn-1, sunn-4, and lss plants compared with the ethylene-insensitive skl mutant. Three plants of each genotype per sample were measured at 10 d post inoculation, with three biological replicates each. Error bars indicate se.

Genetic Interaction between sunn and lss Mutants

To test the allelism of lss with other published supernodulation mutants, we performed genetic crosses. A cross of lss and skl1-1 (Penmetsa et al., 2008) resulted in five F1 plants showing wild-type nodulation and root length; this indicates that lss is not allelic to skl. To test the allelism of lss and sunn, we performed crosses to the sunn-1, sunn-2, and sunn-4 mutants. As previously described, the sunn-1 allele is a missense mutation in the kinase domain, sunn-2 is a missense mutation in a residue between two Leu-rich repeats, and sunn-4 is a truncated allele created by a nonsense mutation before the Leu-rich repeat region (Schnabel et al., 2005). The phenotypic difference between the sunn mutants and lss in nodule number is quantitative and varies slightly between growth conditions; the F1 plants did not consistently show nodule number matching a single parental phenotype, but all F1 plants were supernodulators. The noncomplementation of lss and sunn alleles could be interpreted as evidence that sunn and lss are lesions in the same gene. Reciprocal crosses to lss were performed with the sunn-1 allele. The phenotypes of the F1 generation of the reciprocal cross resembled those of the original cross, indicating that the noncomplementation was not due to parental imprinting.

The lss and sunn Mutations Map to the Same Region

A mapping cross of lss to the A20 ecotype yielded 3,662 F2 progeny, 692 of which displayed the lss phenotype. Using publicly available cleaved-amplified polymorphic sequence (CAPS) markers and some developed for this work (see “Materials and Methods”), we were able to identify recombination events in plants bearing the lss mutant phenotype that place the lss lesion on the lower arm of linkage group 4 near the marker DNABP on the M. truncatula linkage map (Fig. 4A). Markers between the ES10P20-2 and AJ16C13B CAPS markers displayed the homozygous A17 genotype on all lss homozygotes. Combined with data from the M. truncatula sequencing project, the recombination events allowed construction of a contig of sequenced bacterial artificial chromosomes (BACs) defining a segment of linkage group 4 of approximately 810 kb in length containing the lss mutation. Only the 61L2 BAC in the contig is not sequenced, but end sequences place it in a bridging position between 78C5 and 167K21. The SUNN gene is within this 810-kb region, as well as 174 other genes in this segment identified by the International Medicago Gene Annotation Group. Analysis of the International Medicago Gene Annotation Group annotation reveals that almost 20% of the sequence in this contig is annotated as retroelements and repetitive sequence elements. The recombination rate in the lss × A20 population is approximately half the rate in the sunn-1 × A20 population, which we had previously identified as suppressed in recombination (Schnabel et al., 2003), and the suppressed region extends over a much larger region than observed in the original data mapping SUNN.

The region surrounding the lss lesion on linkage group 4. A, Contig containing the lss lesion with supporting BACs and location of recombination events from an F2 mapping population of A20 crossed to lss. DNABP (red) is a publicly available marker (http://www.medicago.org/genome/map_chr.php?chr=4). PCR markers shown above the chromosome line are based on sequence polymorphisms between A17 and A20 genotypes (see “Materials and Methods”). BAC numbers in the contig below the chromosome line refer to the Mt_ABb library. The 61L2 BAC is shown as a dashed line because it has only been end sequenced, so the length and sequence contents are uncertain. The region inside the red box is expanded in B. B, The region of the genome immediately surrounding the SUNN gene in A. Numbers refer to length in bases. Regions in blue have been completely sequenced and shown to be wild type in lss plants.

To confirm that lss was not a mutation in SUNN, in the linked gene RLP1 (Schnabel et al., 2005), or in regulatory regions surrounding SUNN, we sequenced the SUNN region in lss. The sequenced DNA included the SUNN gene as well as the neighboring RLP1 gene (Schnabel et al., 2005), the entire 6.4-kb region between SUNN and RLP1, and 7.2 kb past the SUNN 3′ untranslated region to the start of the next gene in lss plants. We found no DNA base changes in the sequenced regions from lss plants compared with wild-type plants (Fig. 4B).

The Expression of SUNN Is Altered in the lss Mutant

Since the phenotypes of lss and sunn-1 mutant plants are most different from each other, we compared SUNN expression in sunn-1 and lss plants. Previous work determined that the expression level of the SUNN gene in sunn-1 plants was wild-type level in the shoots and reduced to about one-third of wild-type level in the roots (Schnabel et al., 2005). Analysis of SUNN expression levels in lss plants by real-time reverse transcription quantitative PCR (RT-qPCR) showed a similar reduction of SUNN message in lss roots (Fig. 5A) but an almost complete absence of SUNN expression in lss shoots (Fig. 5B). This unexpected result is discussed below. Plants heterozygous for the lss mutation, which have a wild-type nodulation phenotype, display an intermediate level of SUNN expression in the shoots (Fig. 5, C and D) and almost wild-type levels in the roots. In this second set of biological replicates, SUNN expression was undetectable in any tissue of the lss homozygote, but the zero value is within the range of error in Figure 5 (A and B).

SUNN gene expression. A and B, Real-time RT-qPCR of relative SUNN expression levels in roots (A) and shoots (B) of wild-type, sunn-1, and lss plants. Error bars indicate se of relative expression. Expression has been normalized to 1 in wild-type roots and shoots for comparison (see “Materials and Methods”). C and D, Real-time RT-qPCR of relative SUNN expression levels in roots (C) and shoots (D) of plants heterozygous for the lss mutation. The zero value for SUNN expression in lss in C and D is within the range of error in A and B. In all experiments, SECRET AGENT (Kuppusamy et al., 2004) was used as the RT-qPCR control.

Molecular Basis of Reduced SUNN Expression in lss Shoots

We reasoned that the lack of shoot expression of SUNN in lss plants could be due to a cis-element that affects SUNN expression. If so, plants heterozygous for lss would have reduced SUNN expression, because the chromosome containing the lss lesion would not express the SUNN gene. To investigate this possibility, we created plants heterozygous for lss and the sunn-2 mutation and for lss and the sunn-1 mutation. In these plants, one copy of linkage group 4 carries a wild-type allele of SUNN and a mutant allele of LSS, while the other carries a mutant allele of SUNN (sunn-2 or sunn-1, from which the messages can be distinguished from the wild type by restriction analysis of a PCR product from the cDNA) and a wild-type copy of LSS (Fig. 6A). This resulted in plants with the following genotypes: SUNN lss/sunn-1 LSS and SUNN lss/sunn-2 LSS. Analysis of cDNA generated from the shoots of these heterozygotes demonstrated that only the mutant sunn-1 and sunn-2 alleles (the ones on the same chromosome as wild-type LSS) were predominantly expressed in the heterozygote (Fig. 6B). Wild-type SUNN gene expression from the SUNN lss chromosome was very low to nonexistent, producing only a faint band (Fig. 6B, sunn-1 gel) or no band (Fig. 6B, sunn-2 gel).

Origin of expressed SUNN in sunn LSS/lss SUNN plants. A, Diagram of mutation and PCR product locations and restriction sites used in B. H indicates a HaeIII restriction site, while B indicates a BsmAI site; within the PCR product, differential digestion is expected from the two alleles, giving bands of the sizes indicated in the diagram. wt, Wild type. B, Gel electrophoresis of PCR products generated by amplification of cDNA isolated from shoots of the indicated plants, with SUNN-specific primers, followed by digestion with the restriction enzyme HaeIII or BsmAI (see A and “Materials and Methods”). Digestion products from two heterozygotes (A and B) of lss and sunn-1 are shown on the gel at left, and products from lss and sunn-2 are shown at right. Note that in both cases, a band under 100 bases is also generated; these have been left off the image for simplicity, as the sizes of the major bands are diagnostic. MW, Molecular weight in bp. C, Real-time RT-qPCR of relative SUNN expression levels in shoots of A17, lss, and two F3 progeny of a lss revertant. The F2 parent plant segregated only wild-type plants, indicating that it was now homozygous for the reversion event.

Reversion Events

Over the course of the work described here, we noticed occasional revertants, progeny from lss parents that exhibited a wild-type root and nodulation phenotype. We wondered if these reversion events restored SUNN expression to normal levels. In two separate experiments, three revertants were isolated from a total of 600 lss homozygotes grown from two independent batches of seed. The three revertants displayed a wild-type level of nodulation and had longer roots compared with the supernodulators. Selfing of the revertants and phenotypic analysis of their progeny produced segregation of a wild-type level of nodulation in a 3:1 ratio with supernodulators (χ2 = 0.201, P < 0.05), suggesting that one copy of the lss lesion was still present. We identified an F2 offspring of a revertant that segregated only wild-type plants in the F3 generation. We used this small sample of plants presumably homozygous for the reversion event to ask if these reversion events restored SUNN expression to normal levels. Examination of the expression level of the SUNN gene in the shoots of two F3 individuals by RT-qPCR showed a return to normal levels of SUNN expression (Fig. 6C).

In a phenotypic analysis of F2 plants from the selfed F1 generation of the sunn-1 lss cross, 29 plants from a total of 308 individuals had wild-type nodulation. The remaining 279 plants displayed a supernodulation phenotype. This result is unexpected if the two mutations are in the same gene, but because of the apparent close linkage of the two mutations, it is also highly unlikely that these wild-type plants represent recombination events between two linked genes. It remains possible that these wild-type plants are reversions of the lss phenotype.

Methylation Analysis

We wondered if changes in methylation of the SUNN promoter could explain the origin of the low expression of SUNN in lss plants, the observed reversion events, and the wild-type progeny observed in the F2 plants resulting from a cross between sunn and lss plants. We analyzed the methylation status of the SUNN promoter in young leaves of wild-type and lss plants (the same tissue type used in Fig. 6) by bisulfite genomic sequencing. Four overlapping fragments selectively amplified from the lower strand of the bisulfite-converted DNA of the 1.3-kb region upstream of the start codon (−1,295 to −67) were used to evaluate the overall methylation status of the SUNN promoter. The resulting data represent approximately half of the Cs in the SUNN promoter region and include 11 CG, nine CHG, and 146 CHH sites, where H may be A, T, or C (Fig. 7A).

Methylation analysis of the SUNN promoter in wild-type and lss plants. A, Percentage of modified Cs in the SUNN promoter in wild-type A17 (top) and lss mutant (bottom) plants in DNA from shoot tissue. Each tick mark across the x axis denotes a C in the lower strand sequence. The y axis shows the percentage of time that C was methylated in the sequencing of 10 clones. Some variation is expected due to the size of the sample. The bracket indicates an area of interest evaluated in B. The diagram below shows the position of the MITE and the bracketed region relative to the x axis (see text). B, Results of bisulfite sequencing of the area of interest from A in matched tissue from wild-type (A17; dark gray), lss (medium gray), and lss revertant (light gray) plants in two independent experiments. The bases indicated by number are either the bases delineating the region (in A) or instances in which a consistent pattern of small differences in methylation was seen (in B), but always biased toward greater methylation in plants expressing SUNN. Base numbers refer to GenBank accession number AY769943.

The pattern of DNA methylation in this region did not differ dramatically between the wild type and lss. In the region −800 to −67, which includes the predicted transcription start site, little to no methylation was detected. In contrast, within the predicted 184-bp miniature inverted repeat transposable element (MITE) located at positions −1,333 to −1,150, methylation was predominant, and methylation was common in the flanking 300 to 400 bases in all contexts (CG, CHG, CHH).

One small area downstream of the MITE displayed minor differences in methylation between wild-type and lss plants in our experiment (Fig. 7A, bracket). In each case, within the region, methylation was slightly lower in lss plants than in wild-type or revertant plants. To ascertain if these slight differences were contributing to the reduced SUNN expression we observed in lss plants, we followed up with bisulfite genomic sequencing from young leaves of a lss revertant and a second round of bisulfite genomic sequencing on DNA isolated from leaves of younger wild-type, lss, and lss revertant plants (Fig. 7B). Both experiments used types of tissue in which differential expression of SUNN between wild-type and lss plants had been confirmed. This allowed us to observe the variability present in our bisulfite sequencing assay as well as to compare the three plant lines. In most cases in which a small difference in methylation was detected, it was within the noise of the experiment and did not consistently occur between experiments. In five cases indicated by base numbers internal to the graph axis in Figure 7B, a base was more often methylated in wild-type and lss revertant plants than in lss plants by 10% to 30% in both experiments. Analysis of the SUNN promoter at the PLACE database (www.dna.affrc.go.jp/PLACE; Higo et al., 1999) did not identify these bases as part of any recognized promoter motif.

DISCUSSION

The lss mutation in M. truncatula is named for its similarity to the sunn mutation when the phenotypes of nodule number and root length are compared (Figs. 1 and 2) and because of the shoot control of the supernodulation phenotype (Table I). It also maps to the same region of the genome as the sunn mutation (Fig. 4) and does not rescue the sunn mutation when heterozygous. However, the lss mutant differs from sunn-1 in nodulation regulation by nitrate (Fig. 1A), nodulation sensitivity to ethylene (Fig. 3), and gene expression phenotypes (Fig. 5); in these cases, lss behaves like the sunn-4 presumed null allele. These data and the lack of any DNA lesions in the SUNN gene and the surrounding area in lss mutants imply that lss is a novel recessive mutation in M. truncatula that results in a supernodulation phenotype due to insufficient SUNN expression. The observation of putative reversion events suggests an epigenetic effect, but this effect is not through increased methylation of the promoter of the SUNN gene (Fig. 7).

With the exception of the astray mutant and the root-controlled rdh1 and skl mutants discussed in the introduction, all published supernodulation mutants show at least partial nitrate-tolerant nodulation, including the sunn mutant (Jacobsen and Feenstra, 1984; Carroll et al., 1985a, 1985b; Park and Buttery, 1989; Sagan and Duc, 1996; Wopereis et al., 2000; Oka-Kira et al., 2005; Schnabel et al., 2005; Magori et al., 2009). However, we saw differential response to nitrate when comparing lss, sunn-4, and sunn-1 nodulation on NH4NO3 or KNO3 (Fig. 1B). The sunn-4 and lss mutants continued to hypernodulate in the presence of nitrate, while sunn-1 nodule number was significantly reduced in both KNO3 and NH4NO3 compared with no-nitrate conditions. This suggests that sunn-1 mutants sense and respond to the presence of nitrate differently than sunn-4 and lss plants, which continue to supernodulate in the presence of both KNO3 and NH4NO3. Both sunn-4 and lss presumably have little or no SUNN protein, implying that the sensing or response mechanism for nitrate regulation of nodulation requires SUNN.

Nitrate has been shown to inhibit nodulation by the induction of ethylene and by altering levels of flavonoids, auxins, and cytokinins (Coronado et al., 1995; Caba et al., 1998; Mathesius et al., 2000). Here, we found differences in ethylene response between the sunn-1 allele and lss. The sunn-4 and lss plants are more phenotypically similar to wild-type plants than sunn-1 plants, being less sensitive to ethylene’s effects on nodulation (Fig. 3, A and B), yet they all produce similar amounts of ethylene on a per gram fresh weight basis (Fig. 3C). Increasing ethylene by adding ACC or decreasing ethylene by adding AVG (Fig. 3, A and B) reveals a differential response between sunn-1 compared with lss, sunn-4, and the wild type over a range of ethylene concentrations.

Because of the dramatic drop in SUNN expression in the shoots of lss plants (Fig. 5), we speculate that the phenotypic differences above result from insufficient SUNN expression in lss plants versus the expression of a mutant protein in sunn-1 plants. The sunn-4 mutant, which is presumed to lack SUNN protein, phenocopies lss in nitrate response (Fig. 1) and ethylene response (Fig. 3), suggesting that reduced SUNN expression causes the lss phenotype.

The lack of complementation in sunn LSS/SUNN lss plants can be explained by the reduced level of expression of the wild-type copy of the SUNN gene in lss heterozygotes (Fig. 4D) and is further supported by the observation that the sunn-1 mutant has a hemizygous phenotype (Penmetsa et al., 2003), suggesting a sensitivity to gene dosage. Intergenic noncomplementation as seen with lss and sunn can occur with alleles from the same gene when dosage is important, but it can also occur when two separate genes are acting in series or parallel in a signal cascade that is sensitive to gene dosage (Hawley and Walker, 2003). This result is consistent with the SUNN kinase signal being titrated or with SUNN acting in a multimeric complex.

Molecular and genetic tests show that lss is a cis-acting element that affects SUNN expression without affecting SUNN sequence: the SUNN gene itself is wild type but is poorly expressed from the lss chromosome in the sunn LSS/SUNN lss heterozygote (Fig. 6B). The identification of revertants in which the level of SUNN expression is essentially restored to wild-type levels suggests that an unstable or epigenetic mechanism may be operating to produce the phenotype. A simple explanation for the wild-type plants that appear in the F2 generation of the cross between sunn and lss may be reversion of this epigenetic effect. Analysis of lss RNA on the Affymetrix 50K GeneChip Medicago Genome Array Microarray showed that SUNN was the only gene on the chip with altered expression in the area genetically defined to contain LSS (data not shown), suggesting that whatever the lss lesion is, its effect is specific to SUNN.

The lack of dramatic differences in methylation in the SUNN promoter region between lss and wild-type plants implies that promoter methylation is not a likely source of the silencing. Methylation was high near the MITE, a common occurrence in plants (Rabinowicz et al., 2003), and low to nonexistent at the transcript start site, which is common even to plant genes that undergo imprinting (Gehring et al., 2009). We also identified five bases with consistently differential methylation in lss compared with wild-type genomic DNA. However, the differences are small (10%–30%), and while they correlate with expression levels, the direction of the correlation is opposite to known methylation-directed control of plant gene expression. In all five cases, methylation was higher in wild-type and lss revertant plants that express SUNN than in lss, which expresses little SUNN. These latter considerations suggest to us that the small differences detected do not account for the reduction of SUNN expression in lss.

While we cannot yet rule out methylation changes elsewhere in the gene, the majority of methylation events affecting transcription in plants have been reported to occur in the promoter, within 2 kb of the gene start (Penterman et al., 2007), and methylation of exons is much rarer in plants than in mammals (Rabinowicz et al., 2003). Other possible epigenetic modifications influencing expression, such as histone modifications, will require more study. Histone methylation has been shown to play a critical role in the proper expression of some genes in plant development and is a major silencing mechanism in Arabidopsis (Vaillant and Paszkowski, 2007, Zhang et al., 2007b). It is possible that the lss lesion could be the loss of an insulator element, allowing histone modification to spread across SUNN. Alternatively, lss could be the loss of an enhancer element required for SUNN expression or a change in the histone modifications at this element. In these cases, reversion events would occur when methylation failed to reach the required threshold for stable maintenance. However, loss of insulator or enhancer elements would have to occur outside the area already sequenced.

Knowing the identity of lss will be helpful to devising a model of the regulation of nodule number. If LSS is a DNA lesion with epigenetic effects on chromatin structure, precise localization may be difficult. The map-based cloning of SUNN revealed that recombination is suppressed in this area of the genome (Schnabel et al., 2003), and comparison of the genetic and physical distance in the lss mapping population confirms this. The recombination rate in the lss × A20 population is 50% lower than the suppressed level in the sunn-1 × A20 population; our data now show that the suppressed region extends over a larger region than we originally observed when mapping SUNN. The lss lesion is in the Jemalong cultivar, while the sunn mutation is in the A17 genotype, which was derived from the Jemalong cultivar in M. truncatula and is the source genotype for genomic sequencing. It may be that genomic differences, including an unknown genome rearrangement between Jemalong and A17, might explain the suppression of recombination rate. Screening almost 3,700 progeny exhausted the mapping population without narrowing the region. For further analysis, we have undertaken a new cross to the DZA315 genotype to overcome the apparent limitations of the lss × A17 crosses.

In summary, our data indicate that lss regulates nodule number through the modification of SUNN expression. While lss mutants are similar to all sunn mutants on a gross morphological level, plants carrying the lss mutation display nodulation less sensitive to nitrate and ethylene than sunn-1 plants, phenotypically copying the sunn-4 presumed null allele. The reduced expression of the SUNN gene in the shoots of lss plants and in lss heterozygotes, and the lack of complementation of lss and sunn when heterozygous, suggest that the main effect of the lss lesion is to reduce SUNN expression. Analysis of cDNA from plants heterozygous for sunn and lss suggests that lss is a cis-acting factor that inhibits the expression of SUNN. Reversion of this lack of expression occurs naturally, but lack of expression is not correlated with an increase in promoter methylation. Thus, lss represents either a distal novel locus within the mapped region affecting SUNN expression or an uncharacterized epigenetic modification at the SUNN locus.

MATERIALS AND METHODS

Plant Material

The seeds of all genotypes and ecotypes of Medicago truncatula used were scarified in concentrated sulfuric acid (American Chemical Society 93%–98%) by vortexing for 8 min in 15- or 50-mL sterile plastic tubes, depending on the quantity of seed treated. The seeds were washed in distilled water five times and rinsed once in 50% commercial bleach (6% sodium hypochlorite) followed by a final wash with distilled water five times. Treated seeds were imbibed in water shaking at 50 rpm for 3 to 4 h at room temperature. Following imbibition, the seeds were plated by suspending over sterile water, via surface tension, on the lids of sterile 15-mL petri dishes and were vernalized for at least 2 d in the dark at 4°C before being germinated in darkness at 25°C for 24 h. Germinated seedlings were grown on plates, in aeroponic chambers, or in soil as appropriate to a given experiment.

Plants used for phenotypic analysis and comparison, gene expression, and mapping purposes were grown in an aeroponic chamber on nodulation medium as described by Penmetsa and Cook (1997) with the exception of experiments testing nitrogen tolerance, in which 10 mm NH4NO3 or 10 mm KNO3 was added to the medium. Plants were inoculated with wild-type Sinorhizobium medicae strain ABS7 (Bekki et al., 1987) 4 d after they were loaded onto the aeroponic chamber, unless otherwise indicated. This was done by adding to the chamber a solution made from a 25-mL culture that had been grown in tryptone/yeast extract medium containing 15 μg mL−1 tetracycline at 30°C to an optical density at 600 nm of 0.9, spun down, and resuspended in aeroponic medium.

For plate experiments, plants were grown in vertically oriented sterile Bio-Assay Dishes (245 × 245 × 25; Nunc Brand Products) with 250 mL of buffered nodulation medium (Ehrhardt et al., 1992) including 0.1 μm AVG. The plates were sealed with hypoallergenic cloth tape (Kendall), and the lower two-thirds of the plates were wrapped in aluminum foil to decrease light exposure to the roots. The plants were flood inoculated with rhizobia 4 d after they were plated, unless otherwise mentioned. For tests of plants later than 15 d after inoculations, plants were grown in a greenhouse at 20°C to 25°C with natural light supplemented with a 14-h/10-h light/dark cycle; the growth medium was Middleweight Soil Mix 3-B (Fafard), and all plants were watered daily with a 100-fold dilution of water-soluble 20:10:20 Peat-Lite Special fertilizer (Scotts).

Plant Phenotype Scoring

Plants grown on caissons or plates were scored for nodule number and root length 10 d post inoculation with rhizobia, unless otherwise noted. Root length was measured with a ruler from just below the hypocotyl junction to the root tip. Nodules were counted using a dissecting microscope.

Hormonal Treatments and Measurements

A stock solution of 100 mm ACC was prepared in 1 mL of distilled water. The stocks were filter sterilized during preparation. For ACC treatments, AVG was omitted from the growth medium.

For quantification of ethylene production, three biological replicates of aeroponically grown plants were harvested 10 d post inoculation with rhizobia. Three plants of a single genotype were pooled together into a single serum-stopper 24-mL glass vial and incubated for 4 h in the growth chamber at 25°C. Ethylene was quantified by directly injecting a 1-mL sample of the air space from the vial into a gas chromatograph (Shimadzu GC-9A) equipped with a flame photometric detector; column temperature was 30°C and injection temperature was 130°C; supply pressure was as follows: air (50 p.s.i.), H2 (50 p.s.i.), N2 (100 p.s.i.). Empty vials were also sampled in triplicate to account for background ethylene in the air. The fresh weight of the plants was measured, and the amount of ethylene produced was divided by the respective fresh weight.

Genetic Crosses

Artificial hybridization was performed as described by Pathipanawat et al. (1994). Emasculated buds 2 d prior to full anthesis were used for crossing purposes, as they were most amenable to cross-pollination and least prone to self-pollination (Penmetsa and Cook, 2000). Pollen was removed using a hand-held vacuum system from the pollen-recipient parent, and pollen from freshly tripped flowers of the pollen-donor parent was added to the stigma of the emasculated flowers using fine tweezers as described by Penmetsa and Cook (2000). When crossing phenotypically similar individuals, we used a male-sterile floral homeotic mutant of M. truncatula ‘Jemalong’, Mtapetala, to eliminate the possibility of obtaining false positives from our crosses. The apetala mutant did not require emasculation, and the effects of the apetala mutation are restricted only to floral organs; nodulation is indistinguishable from that of the wild type (Penmetsa and Cook, 2000). The resulting pods were wrapped in surgical gauze and labeled to aid collection.

Genetic Mapping

Individuals homozygous for the lss mutation were hand emasculated and crossed with pollen from ecotype A20, as described by Schnabel et al. (2003). The resulting F1 progeny from the lss × A20 cross were allowed to self-pollinate to generate multiple, independent F2 populations. Progeny from eight lss × A20 F1 plants were screened for the lss phenotype, based on the nodule number and root length. Plants assumed homozygous for the lss mutation based on the supernodulation phenotype were used to map the lss mutation by following the segregation of the mutant phenotype with CAPS markers and other molecular markers (Table II), some of which were previously developed against available EST and BAC sequences (Schnabel et al., 2003). DNA from these plants was isolated using DNeasy Plant Kits (Qiagen) and FTA Plant Cards (Whatman). The genomic DNA was used as the template to amplify PCR fragments using primer sets specific for each marker, followed by digestion with the appropriate enzyme. Gel electrophoresis and ethidium bromide staining were used to observe the genotype of the plant at that particular marker. Because each marker is inherited in a codominant fashion, all allelic combinations were readily scored. The resulting genotype assignments were observed and compiled in a color map that consisted of all plants with informative recombination events to visualize the distance between the markers that flank lss (Kiss et al., 1998).

Quantification of Gene Expression

Total RNA was extracted from plant tissues using the Qiagen RNeasy Plant Mini Kit according to the manufacturer’s instructions. RNA was extracted from roots and shoots from 14 d-old-plants (10 d post inoculation) gathered in three independent biological replicates. Approximately 1 to 2 μg of RNA was used as the template for single-strand cDNA synthesis using random hexanucleotide primers (Invitrogen) and SuperScript Reverse Transcriptase II (Invitrogen) following the manufacturer’s instructions. The same amount of RNA was used as starting material for each sample within an experiment.

In previous work (Schnabel et al., 2005), gene-specific intron-spanning primers were designed to amplify a 523-bp fragment from the kinase domain of SUNN cDNA. For all expression studies, the M. truncatula SECRET AGENT gene was used as an internal control, as described by Schnabel et al. (2005). Gene expression levels were quantified by qPCR in a Bio-Rad iCycler. Detection of gene expression levels occurred in 25-μL PCRs consisting of iQ SYBR Green Supermix (Bio-Rad). Cycling conditions were 95°C for 3 min, followed by 40 cycles of 95°C for 10 s, 61°C for 20 s, 72°C for 1 min, and 82°C for 10 s. Fluorescence data (excitation filter, 480/20; emission filter, 530/30) were collected at 82°C to prevent signal detection from primer-dimers from interfering with the signal from the specific PCR products.

PCR threshold cycles (Ct) were determined from PCR baseline-subtracted curve-fit data using default parameters of the iCycler software package. A dilution series of a known template was used for each experiment to calculate the efficiency (E) of the PCR for a given primer pair from the slope of the regression line from a plot of Ct versus log (template concentration) using the formula E = 10(−1/slope). For each primer pair on a biological replicate, three technical replicates were used, and average Ct values were calculated from these values.

Expression in lss/sunn Heterozygotes

The cDNA from F1 plants of lss × sunn-2 or lss × sunn-1 was analyzed with the sunn-1 or sunn-2 marker to determine if the heterozygote expressed either the lssSUNN or the sunn-2 allele, the sunn-1 allele, or both. cDNA from heterozygote plants, A17 wild-type plants, and genomic DNA from A17 and sunn-2 plants were extracted as described. These were used as templates to amplify a 414-bp fragment using primers 5′-AGAATCTGAAGGTTCTAAGCATTTTT-3′ and 5′-GACACTCCACTACTTCCTCTGCT-3′ for sunn-2 and 5′-CATGTTGCTGATTTTGGACTTG-3′ and 5′-CCTGGTACCCTAGAGATTAATCAAGTTGTGACT-3′ for sunn-1. The annealing temperature used was 57°C. Following amplification, the PCR products were digested with BsmAI (New England Biolabs) at 55°C for 4 h, which cuts only the sunn-2 allele, or HaeIII (New England Biolabs) at 37°C for 4 h, which cuts only the sunn-1 allele. Following digestion, the samples were run on a 1% agarose gel and stained with ethidium bromide.

Bisulfite Sequencing

Genomic DNA (500 ng) from leaves of greenhouse-grown plants was converted with bisulfite using the EZ Methylation Direct Kit (Zymo Research) following the manufacturer’s protocol. SUNN promoter fragments were amplified by PCR from bisulfite-converted DNA using primer pairs designed using Kismeth (http://katahdin.mssm.edu/kismeth/primer_design.pl) that are specific for amplification from the lower strand of converted DNA (Gruntman et al., 2008). PCR products were gel purified, extracted using UltraFree DA columns (Millipore), ethanol precipitated, and cloned using the pGEM T-Easy Vector System (Promega). Ten clones were sequenced from each region in each replicate to determine percentage methylation.

Statistical Analysis

Acknowledgments

We thank Anna Marshall, Kelly Jones, and Ryan Fernandez for assistance with the mapping and Melissa Riley for advice and use of equipment in the measurement of ethylene production.

Footnotes

↵1 This work was supported by the National Science Foundation (grant no. IOB–0641848 to J.F.), by a Clemson University Public Service and Agriculture Next Generation Graduate Fellowship to T.K., by the Hoover Circle Fund and prior support from the Howard Hughes Medical Institute, and by the U.S. Department of Energy (grant no. DE–FG03–90ER20010 to S.R.L.). This is technical contribution no. 5764 of the Clemson Experiment Station.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Julia Frugoli (jfrugol{at}clemson.edu).

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